JP7260878B2 - Cell suspension used to treat lower extremity peripheral artery disease - Google Patents

Description

The present invention relates to a cell suspension of adult bone marrow-derived cells that can be used for the treatment or amelioration of lower extremity peripheral artery disease, preferably critical ischemia limb.

Lower extremity peripheral arterial disease (PAD) includes various vascular diseases caused by atherosclerotic, thromboembolic and inflammatory processes that alter the structure and function of the lower extremity arteries. However, the primary cause of PAD is atherosclerosis.

Symptomatic lower extremity PAD results from inadequate blood flow that causes a lack of oxygen supply. PAD is associated with atherothrombosis of other vascular beds, including the cardiovascular and cerebrovascular systems. The presence of diabetes mellitus (DM) greatly increases the risk of PAD and accelerates disease progression, leading to ischemic events, impaired vascular function, increased morbidity and mortality, and reduced quality of life. (QoL) deterioration (Thiruvoipati et al, 2015). In fact, PAD patients are considered a heterogeneous group of patients ranging from asymptomatic to those with intermittent claudication (IC) and those with CLI (Critical Limb Ischemia). CLI refers to a state of arterial insufficiency in which peripheral perfusion pressure drops to the point that microcirculation and blood flow and nutrients to tissues are severely perturbed. Mild trauma, ulceration and infection can occur together, increasing the metabolic demands of the affected tissue and often leading to chronic non-healing ulcers. CLI is a progressive disease characterized by multiple levels of disease, a high burden of comorbidities and a limited lifespan.

The ankle-brachial index (ABI) is used to diagnose PAD and is an indicator of the severity of PAD, but does not perfectly correlate with clinical severity of the disease. Therefore, a broader range of clinical symptoms is used to classify the severity of PAD, and two classification systems, Rutherford and Fontaine, are routinely used in clinical practice (Hirsch AT, 2006). The clinical severity of PAD varies from asymptomatic to gangrene and leg ischemia requiring amputation. PAD is a progressive disease in which the severity of the condition increases over time. Disease progression is more rapid in patients with DM.

The grading of symptoms of PAD and the anatomic lesions associated with these symptoms provides an objective measure for clinical follow-up of subjects and, importantly, guides medical and interventional interventions in clinical studies. Brings consistency over strategy.

The Rutherford classification is a clinical staging system commonly used to describe PAD, similar to the Fontaine classification but commonly cited by more recent publications in the field of vascular medicine. Peripheral arterial occlusive disease is generally classified into Fontaine stages I (asymptomatic) to IV (ulceration or gangrene) in the Fontaine stage introduced in 1954 by Rene Fontaine. The classification, introduced by Rutherford in 1986 and revised in 1997, consists of 4 grades and 7 categories ranging from Group 0 (asymptomatic) to Group 6 (severe ischemic ulcer or gangrene) (Table 1). .

CLI is a progressive disease characterized by multiple levels of disease, a high burden of comorbidities and a limited lifespan. The Trans-Atlantic Inter-Society Consensus (TASC) Document on Management of Peripheral Arterial Disease II, 2007 (TASCII, 2007) defines the term CLI as chronic ischemic resting conditions resulting from objectively documented arterial occlusive disease. It is recommended that it should be used in all subjects with pain, ulcers or gangrene. The current definition of CLI includes subjects with lower extremity rest pain, ulceration or gangrene secondary to severely impaired blood flow in the extremity lasting more than 2 weeks. This corresponds to Rutherford's groups 4-6 (ischemic resting pain, minor and large tissue defects, respectively) or Fontaine's grades III and IV.

The only treatment for the underlying disease in CLI is revascularization. However, a significant proportion of the population (estimated at 50% to 90% of the population) undergoes manual revascularization (estimated at 50% to 90% of the population) due to either lack of viable vessels, failure of previous manual revascularization or comorbidities. Not suitable for bypass surgery or endovascular surgery). Many patients with CLI have multiple medical conditions that may prevent them from undergoing manual revascularization. Ultimately, amputation, aimed at managing intractable pain, preventing gangrene progression, or combating infection, is often the only remaining option for most of these patients. . Therefore, new therapies are urgently needed.

Currently, initial treatment of patients with CLI usually consists of evaluation of the affected (indicator) limb for suitability for revascularization by bypass surgery or using endovascular methods. All patients are treated for existing symptoms of disease, especially pain, ulcers and infections. However, because pain control is so often ineffective and ischemic ulcers rarely heal, wound management is usually focused on infection control, prevention of exacerbation and salvage of the injured limb. Overall treatment goals for CLI include:
Pain control Wound healing Injured limb relief QoL improvement including maintenance of ambulatory status Reduction of overall cardiac risk

The cell suspension of the present invention is particularly indicated for CLI patients, especially CLI patients with DM who do not have the option of bypass surgery or revascularization using endovascular therapy. These patients have the greatest unmet need as there are no therapeutic options to treat the underlying disease. Any treatments used in these patients are symptomatic, such as wound healing, infection control and pain management. For those patients who are not suitable for manual revascularization, as disease progresses, amputation may become the only clinical option, depending on either progressive gangrene, uncontrolled infection, or intractable pain. be. Approximately 25% of PAD patients who develop CLI die within one year (NICE, 2012). Depending on the severity of CLI, the yearly primary amputation rate ranges from 5% to 40%, and is highest in patients who are unsuitable for revascularization, neuropathic or non-ambulatory.

The problem addressed by the present invention is patients suffering from lower extremity peripheral arterial disease (PAD), preferably CLI patients, more preferably CLI patients with DM who do not have the option of bypass surgery or revascularization using endovascular therapy. is to provide improved treatment options for Such improved treatment options are provided by the present invention in the form of a cell suspension of autologous or allogeneic adult bone marrow-derived cells defined by the aspects and preferred embodiments referred to below.

Thus, in a first aspect of the invention, enriched for mononuclear cells (more than 25% of the white blood cells (WBC) present in the cell suspension are mononuclear cells),
A.
i. a cluster of lymphocytes,
ii. a population of monocytes, and
iii. a population of hematopoietic stem cells that express CD34;
comprising a mononuclear cell (MNC) selected from the list comprising or consisting of
B. granulocytes,
A cell suspension of autologous or allogeneic, preferably autologous, adult bone marrow-derived leukocytes, preferably derived from the crest of the ilium (or iliac crest) (hereinafter referred to as "the cells of the present invention suspension”).

In general, the cell populations A) and B) comprise a cell population expressing CXCR4, the receptor for the chemotactic factor SDF1, and pointing out the existence of a subpopulation of hematopoietic stem cells expressing CD34 and CXCR4 from this population. is important. In addition, cell populations A) and B) comprise additional cell populations expressing VEGFR2, the receptor for the angiogenic factor VEGF involved in angiogenesis and vasculogenesis.

Finally, cell population A. iii) comprises a cell population that does not express CD38, the primary non-committed hematopoietic stem cell.

Note that mononuclear cells (MNC) are considered the main active component of the cell suspensions of the present invention. In particular, the active constituents or ingredients of the cell suspension of the invention are lymphocytes, monocytes and hematopoietic stem cells expressing CD34, in particular a subpopulation of early uncommitted hematopoietic stem cells expressing CD34 and not CD38, CXCR4 leukocytes capable of SDF-1-mediated migration expressing CD34 and CXCR4, stem cells capable of SDF-1-mediated migration expressing CD34 and leukocytes capable of angiogenesis expressing VEGFR2. . All of these mononuclear cells are bone marrow-derived cells, preferably derived from the iliac crest.

Additionally, it is further noted that the leukocyte suspension of the present invention further comprises granulocytes that are neutrophils, eosinophils and/or basophils. In this regard, the final cell suspension product of the present invention described in the Examples is based on viable leukocyte bone marrow-derived cells that include not only MNC leukocyte fractions (lymphocytes and monocytes and stem cells), but also granulocytes. It is important to note that the

In a preferred embodiment of the first aspect of the invention, enriched for mononuclear cells (greater than 25% of the total number of white blood cells (WBC) present in the cell suspension are mononuclear cells). The cell suspension of the invention in the final formulation contains about 4×10 ⁸ to about 2×10 ⁹ autologous or allogeneic, preferably autologous, cells, preferably derived from the iliac crest. Of the total number of leukocytes from about 4×10 ⁸ to about 2×10 ⁹ , including leukocyte bone marrow-derived cells,
i. about 20% to about 51% are lymphocytes and about 3.9% to about 22.3% are monocytes;
ii. about 1.4% to about 10% are hematopoietic stem cells that express CD34;
iii. About 25.3% to about 83.3%, preferably about 32.3% to about 80.0%, of the total number of white blood cells are preferably single cells selected from the list consisting of lymphocytes, monocytes and CD34 cells. is a nuclear cell,
iv. about 16.7% to about 74.7%, preferably about 20.0% to about 67.7% are granulocytes;
v. from about 7.7% to about 55.5% of the total number of hematopoietic stem cells that express CD34 are early uncommitted hematopoietic stem cells that do not express CD38;
vi. about 5.4% to about 38.8% of white blood cells express CXCR4;
vii. Of the total number of hematopoietic stem cells that express CD34, about 0.7% to about 10.3% are stem cells that express CD34 and CXCR4;
viii. from about 0.07% to about 24.7% of white blood cells express VEGFR2;
ix. The maximum red blood cell to leukocyte cell ratio is 6.7 and the maximum platelet to leukocyte cell ratio is 32.

In another preferred embodiment of the first aspect of the invention, the cell suspension of the invention in the final formulation defined in the preceding paragraph contains about 4 x 10 ⁸ to about 1.2 x 10 ⁹ white blood cells, more preferably from about 5×10 ⁸ to about 2×10 ⁹ white blood cells, even more preferably from about 5×10 ⁸ to about 1.2×10 ⁹ white blood cells, and Include all dose values between each value.

Certain embodiments can be selected as subranges from these values for white blood cells in the final formulation. For example, certain embodiments can be selected with a leukocyte content in the final formulation of greater than 8×10 ⁸ to about 1.2×10 ⁹ leukocytes. Another example of a range that can be selected in embodiments is selecting a white blood cell content of about 9×10 ⁸ to about 1.1×10 ⁹ . A third example of a range that can be selected for a particular embodiment is the selection of about 9.5×10 ⁸ to about 1.05×10 ⁹ white blood cells. A fourth example of a range that can be selected for certain embodiments is the selection of a content such as greater than 9.8×10 ⁸ to about 1.02×10 ⁹ white blood cells.

Another example of a range of content or dosed embodiments that can be selected is from greater than 6 x ¹⁰⁸ to about 2 x ¹⁰⁹ , preferably to about 1.2 x ¹⁰⁹ . , including all dosage values therebetween. Another example is the selection of a range of white blood cells from greater than 7×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 8×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 8.5 x ¹⁰⁸ to about 2 x ¹⁰⁹ , preferably to about 1.2 x ¹⁰⁹ , including all dosage values therebetween. is. Another example is the selection of a range of white blood cells from greater than 9×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 9.5 x ¹⁰⁸ to about 2 x ¹⁰⁹ , preferably to about 1.2 x ¹⁰⁹ , including all dosage values therebetween. is. Another example is the selection of a range of white blood cells from greater than 7×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Thus, all dosage ranges that can be selected from the values herein as understood by one of ordinary skill in the art are encompassed by the present invention.

Methods for measuring each of the above cell populations will be clearly established in the detailed description of the invention.

Note that such cell suspensions may contain pharmaceutically acceptable carriers and/or excipients.

In another preferred embodiment of the first aspect of the present invention, or in any of its preferred embodiments, the lower extremity peripheral arterial disease is critical lower extremity ischemia.

In another preferred embodiment of the first aspect of the present invention or any of its preferred embodiments, the cells are preferably treated with heparin in a volume of 5 ml to 30 ml containing about 1% HSA and about 2.5% glucose. Suspend in added saline or lactated Ringer's solution.

In a second aspect of the present invention, an inflatable balloon is placed proximal to an occlusive vascular lesion in the distal femoral or popliteal artery and the cell suspension is injected intra-arterially to treat hypotension of up to 4 atmospheres. A cell suspension as defined in the first aspect of the invention or any of its preferred embodiments for use in a method of treating lower extremity peripheral arterial disease, preferably critical lower extremity ischemia, by intra-arterial administration to obtain flow. It refers to turbidity.

The third aspect of the invention refers to one or several pre-filled syringes containing a cell suspension as defined in the first aspect of the invention or any of the preferred embodiments thereof.

The fourth aspect of the invention refers to the syringe defined in the third aspect of the invention (pre-filled syringe) for use in the treatment or amelioration of lower extremity peripheral arterial disease, preferably critical lower extremity ischemia. Note that more than one prefilled syringe may be used per patient.

In a fifth aspect of the present invention, an inflatable balloon is placed proximal to the occlusive vascular lesion in the distal femoral or popliteal artery and the cell suspension is injected into the artery to treat hypotension up to 4 atmospheres. Reference is made to the syringe defined in the third aspect of the invention (pre-filled syringe) for use in a method of treating lower extremity peripheral arterial disease, preferably critical lower extremity ischemia, by intra-arterial administration to obtain flow.

In preferred embodiments of the first to fifth aspects of the invention, the cell suspension or syringe is provided as a single dose.

In a preferred embodiment of the second and fifth aspects of the invention, the induction of low pressure flow is for 1 to 6 minutes and the injection of said cell suspension is for 2 to 10 minutes.

In a sixth aspect of the invention, reference is made to a method for producing a cell suspension of the invention as defined in the first aspect of the invention or any of its preferred embodiments, comprising:
1. Bone marrow (BM) harvesting is performed by repeated punctures, preferably from the subject's posterior iliac crest under local or general anesthesia. The BM puncture fluid is then collected in a shipping bag, preferably containing an anticoagulant solution, more preferably an acid citrate dextrose solution A (ACD-A).
2. The BM is preferably filtered by gravity to remove all small bone fragments and prevent blockage in subsequent steps.
3. The initial BM volume is preferably reduced to about 50-100 mL, preferably by using the SmartRedux program with the Sepax 2.0 device and the accompanying sterile disposable SmartRedux kit (CS-490.1). This step, including removal of plasma and red blood cells (RBCs), which preferably yields a buffy coat product of about 50-100 mL, also contributes to the purity of the final product.
4. Optionally, the volume reduction step is performed in one or two cycles depending on the volume of starting material to be processed. BM samples up to 220 mL in volume are processed in a single cycle, and samples over 220 mL are processed in two cycles using the same kit. It is preferred to set the final volume to about 50 mL to 100 mL for both single and two cycle volume reduction.
5. The sterile intermediate sample bag containing the reduced volume BM established in step 3 or step 4 is subjected to automated density gradient centrifugation followed by 2%-4% HSA, preferably in saline solution (both pharmaceutical Two washes of the mononuclear cell (MNC) suspension are performed with a wash solution consisting of: Collect approximately 45 mL of BM-MNC product into an output bag and remove other components into a waste bag. Preferably, the sterile intermediate sample bag containing the reduced BM is subjected to NeatCell density gradient centrifugation followed by preferably consisting of 2%-4% HSA (both pharmaceutical grade) in saline solution. Two washes of the mononuclear cell (MNC) suspension with wash solution are performed. Collect approximately 45 mL of BM-MNC product into an output bag and remove other components into a waste bag.
6. BM-MNC are preferably taken from the product bag using a 50 mL syringe and filtered preferably through a 50 μm filter, preferably into a sterile Falcon tube. Rinse the product bag with a wash solution and filter to improve MNC recovery. Adjust final volume of BM-MNC drug substance to 40 mL-60 mL.

The drug substance is centrifuged and the pellet reconstituted in a volume of 5 ml to 30 ml of the final formulation mixture, preferably saline containing heparin, or lactated Ringer's solution containing preferably 1% HSA and preferably 2.5% glucose. Suspend.

It should be noted that, as previously mentioned above, the final cell suspension product obtained according to the sixth aspect of the invention is administered on the basis of viable leukocytes containing granulocytes as well as the MNC fraction. Since MNC is the active ingredient, the percentage of MNC in the final product is considered a quality attribute.

In a seventh aspect of the invention reference is made to a cell suspension product or product container, preferably one or several syringes, obtained or obtainable by the method of the sixth aspect of the invention. It should be noted that such cell suspension products or product containers can be used as established in any of the first to fifth aspects of the invention.

1 is a flow chart of manufacturing a cell suspension of the present invention. FIG. 3 shows the Rutherford classification (control vs. treatment; including amputation and death; LOCF) after 3, 6, 9 and 12 months of administration of a cell suspension of the invention. FIG. 3 shows the Rutherford Classification (effects of 3 escalating doses; including amputation and death; LOCF) after 3 months, 6 months, 9 months and 12 months after administration of a cell suspension of the invention. . FIG. 3 shows the improvement in the Rutherford classification (percentage of subjects) at 3 months, 6 months, 9 months and 12 months for the control group and the three groups (doses) of the cell suspension of the invention. FIG. 2 shows the percentage of subjects classified by the number of ulcers in the affected limb at 3 months, 6 months, 9 months and 12 months (left panel: control group; right panel: cell suspension of the invention). group). Angiographic images of 3 of the 24 angiogenesis events that occurred are presented relative to baseline in FIG. Parallel lateral growth (Fig. 6C). 1 shows a flow diagram of a method for producing a cell suspension product of the present invention; FIG.

DEFINITIONS As used herein, "autologous" is understood to refer to a cell preparation in which the donor and recipient are the same individual.

As used herein, "allogeneic" is understood to refer to a cell preparation in which the donor and recipient are not the same individual.

As used herein, "adult bone marrow-derived cells" are understood as preparations containing cells derived from bone marrow obtained from non-embryonic, human donors.

As used herein, a "cell suspension" is understood as a preparation of cells suspended in liquid medium.

As used herein, a "cell suspension of adult bone marrow-derived cells" is understood as a preparation of non-embryonic, bone marrow-derived cells obtained from a human donor suspended in a liquid medium. be done.

As used herein, a "CD34-expressing hematopoietic stem cell" is understood as a hematopoietic stem cell that expresses the surface marker CD34 and is identified as CD34-positive by CD34 antibody staining and flow cytometry.

As used herein, "primary uncommitted hematopoietic stem cells that express CD34 and not CD38" express the surface marker CD34 and are identified as CD34 positive by CD34 antibody staining and flow cytometry, It is understood as hematopoietic stem cells identified as negative for the surface marker CD38 by CD38 antibody staining and flow cytometry.

As used herein, "CXCR4-expressing leukocytes" are understood as cells that express the surface markers CD45 and CXCR4 and are identified as positive for CD45 and CXCR4 by CD45 and CXCR4 antibody staining and flow cytometry. be. CXCR4 is the receptor for the migration factor SDF-1.

As used herein, "CD34 and CXCR4 expressing stem cells" express the surface markers CD45, CD34 and CXCR4 and are positive for CD45, CD34 and CXCR4 by CD45, CD34 and CXCR4 antibody staining and flow cytometry. is understood as a cell identified as CXCR4 is the receptor for the migration factor SDF-1.

As used herein, a "VEGFR2-expressing leukocyte" is understood as a cell that expresses the surface markers CD45 and VEGFR2 and is identified as positive for CD45 and VEGFR2 by CD45 and VEGFR2 antibody staining and flow cytometry. be. VEGFR2 is the receptor for VEGF, an angiogenic and angiogenic factor.

As used herein, a "mononuclear cell" is understood as any blood or bone marrow leukocyte (also called leukocyte) with a round nucleus, thus excluding granulocytes.

As used herein, "peripheral arterial disease of the lower extremities" is understood as narrowing of arteries other than those that supply the heart or brain. Peripheral arterial disease commonly affects the extremities, but other arteries can be affected.

As used herein, “critical limb ischemia” is chronic ischemic at-rest pain in one or both legs due to objectively documented arterial occlusive disease, It is understood as a subdivision of peripheral arterial disease characterized by ulceration or gangrene.

The term "about" in reference to a numerical value means ±20% of that numerical value. The term "about" in reference to a number also includes ±10% of that number. The term "about" in reference to a number also includes ±5% of that number. The term "about" in relation to a number also includes ±1% of that number.

The terms "comprise" and "comprising" are used in an inclusive and open sense, meaning that additional elements may be included. The term "comprising" also encompasses, and is sometimes used interchangeably with, the terms "consisting of" and "consisting essentially of."

As used herein, the term "adult" means that the stem cells are non-embryonic. In one embodiment, "adult" refers to the post-embryonic stage or "postnatal." With respect to the stem cells of the present invention, the term "adult stem cell" means that stem cells are isolated from a tissue or organ of an animal at a stage of development after embryogenesis. Adult stem cells differ from embryonic stem cells, which are defined by their origin as the inner cell mass of the blastocyst. Adult stem cells according to the invention can be isolated from any non-embryonic tissue, including neonatal, pediatric, adolescent and adult subjects. Generally, the stem cells of the invention are isolated from a non-neonatal mammal, eg, a non-neonatal human. The stem cells of the invention are preferably isolated from humans.

The term "isolated" indicates that the cell or cell population to which it refers is not in its natural environment. The cell or cell population is substantially separated from surrounding tissue.

The marker profile of the new cell suspension product referred to in the present invention can be further defined by a specific profile of the presence and/or absence of additional markers or a combination of presence and absence markers. . In either case, specific combinations of markers may exist as specific profiles within a population of cells and/or specific profiles of markers on individual cells within a population.

As used herein, the term "marker" includes any biomolecule whose presence, concentration, activity or phosphorylation state can be detected and used to identify the phenotype of a cell.

In this case, the cells of the invention are positive for certain phenotypic markers and negative for other markers. By "positive" is meant that the marker is expressed within the cell. To be considered expressed, a marker must be present at detectable levels. By "detectable level" is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or flow cytometric analysis as described.

The term "expressed" is used to describe the presence of a marker on the surface of a cell or within a cell. To be considered expressed, a marker must be present at detectable levels. By "detectable level" is meant that the marker can be detected using one of standard laboratory methodologies such as PCR, blotting, immunofluorescence, ELISA or FACS analysis. "Expressed" may refer to, but is not limited to, the detectable presence of a protein, the phosphorylation state of the protein, or the mRNA that encodes the protein. A gene is present if its expression can be sufficiently detected after 30 PCR cycles, preferably after 37 PCR cycles (corresponding to an expression level in cells of at least about 100 copies per cell). It is considered to be expressed by the cells of the invention or the cells of the population of the invention. The terms "express" and "expression" have corresponding meanings. At expression levels below this threshold, the marker is not considered expressed.

Detailed Description Cell Suspension Products Cell suspensions according to the present invention are useful options for improved therapy for the treatment or amelioration of lower extremity peripheral arterial disease, especially the treatment of critical lower extremity ischemia (CLI). It is a suitable product for the treatment of critical limb ischemia (CLI) in patients with DM who do not have the option of bypass surgery or revascularization using endovascular therapy. In this sense, surprisingly, when the cell suspension according to the present invention is used in the treatment or amelioration of lower extremity peripheral artery disease, CLI level II (group 4) or level III (group 5) to level I ( A change in the Rutherford classification to 0 degrees (groups 1-3) or 0 (group 0), an improved healing process not only for ulcers but also for major amputations, and increased vessel lengthening and/or vessel density. was found to provide a clinically relevant improvement in All of the above advantages can be obtained even with a single dose of cell suspension and are clearly illustrated in Example 2.

Generally, the cell suspensions of the invention are
A.
i. a cluster of lymphocytes,
ii. a population of monocytes, and
iii. a population of hematopoietic stem cells that express CD34;
comprising a mononuclear cell (MNC) selected from the list comprising or consisting of
B. granulocytes,
autologous or allogeneic, preferably autologous adult bone marrow-derived cells, preferably iliac crest-derived cell suspensions, further comprising

It is important to point out that the cell populations A) and B) comprise a cell population expressing CXCR4, the receptor for the migration factor SDF1, and from this population a subpopulation of hematopoietic stem cells expressing CD34 and CXCR4 exists. Please note. In addition, cell populations A) and B) comprise cell populations expressing VEGFR2, the receptor for the angiogenic factor VEGF involved in angiogenesis and vasculogenesis.

Finally, cell population A. iii) comprises a cell population that does not express CD38, an early uncommitted hematopoietic stem cell.

Thus, in particular, the suspension according to the invention contains i) hematopoietic stem cells expressing CD34, ii) early uncommitted hematopoietic stem cells expressing CD34 and not CD38, iii) receiving the migration factor SDF-1. iv) hematopoietic stem cells expressing CD34 and CXCR4, the receptor for the migration factor SDF-1; v) leukocytes expressing VEGFR2, the receptor for VEGF; and vi) monocytes. A cell suspension of autologous or allogeneic, preferably autologous, adult bone marrow-derived cells, including granulocytes and lymphocytes.

Note that the final cell suspension product of the present invention is administered on the basis of viable leukocyte bone marrow-derived cells, including not only mononuclear leukocyte fractions (lymphocytes and monocytes and stem cells), but also granulocytes. is important.

Thus, the cell suspension of the present invention contains i) hematopoietic stem cells that express CD34, ii) early uncommitted hematopoietic stem cells that express CD34 and do not express CD38, iii) CXCR4, the receptor for the migration factor SDF-1. iv) hematopoietic stem cells expressing CD34 and CXCR4, the receptor for the migration factor SDF-1, v) leukocytes expressing VEGFR2, the receptor for vascular endothelial growth factor (VEGF), and vi ) refers to a cell suspension of autologous or allogeneic, preferably autologous, adult bone marrow-derived cells, including monocytes, granulocytes and lymphocytes.

In a preferred embodiment, the cell suspension in the final formulation is enriched for mononuclear cells (meaning that more than 25% of the white blood cells (WBC) present in the cell suspension are mononuclear cells). teeth,
autologous or allogeneic, preferably autologous, leukocytic bone marrow-derived cells, preferably from the iliac crest, comprising from about 4×10 ⁸ to about 2×10 ⁹ cells, from about 4×10 ⁸ to about 2× Of the total number of 10 ⁹ leukocytes,
i. about 20% to about 51% are lymphocytes and about 3.9% to about 22.3% are monocytes;
ii. 1.4% to 10% are hematopoietic stem cells expressing CD34;
iii. from about 25.3% to about 83.3% of the total number of white blood cells are mononuclear cells;
iv. about 16.7% to about 74.7% are granulocytes;
v. from about 5.4% to about 38.8% of the total number of white blood cells express CXCR4;
vi. from about 0.07% to about 24.7% of the total number of white blood cells express VEGFR2;
vii. Of the total number of CD34-expressing hematopoietic stem cells, about 7.7% to about 55.5% are early uncommitted hematopoietic stem cells that do not express CD38, and about 0.7% to about 10.3% are CD34 and CXCR4. is a stem cell that expresses
viii. The maximum red blood cell to leukocyte cell ratio is 6.7 and the maximum platelet to leukocyte cell ratio is 32.

In another preferred embodiment, 32.3% to 80.0% of the total number of white blood cells are mononuclear cells selected from the list consisting of lymphocytes, monocytes and hematopoietic stem cells expressing CD34;20. 0% to 67.7% are granulocytes.

In yet another preferred embodiment, the cell suspension of the present invention in the final formulation defined in the preceding paragraph contains about 4×10 ⁸ to about 1.2× ¹⁰ 9 white blood cells, more preferably about 5×10 ⁸ to about 2×10 ⁹ white blood cells, even more preferably from about 5×10 ⁸ to about 1.2×10 ⁹ white blood cells, and all between each of the above recited values. Contains dosage values.

Certain embodiments can be selected as subranges from these values for white blood cells in the final formulation. For example, certain embodiments can be selected as having a white blood cell content of greater than 8×10 ⁸ to about 1.2×10 ⁹ white blood cells. Another example of a range that can be selected in embodiments is selecting a white blood cell content of about 9×10 ⁸ to about 1.1×10 ⁹ . A third example of a range that can be selected for certain embodiments is the selection of about 9.5×10 ⁸ to about 1.05×10 ⁹ white blood cells. A fourth example of a range that can be selected for certain embodiments is the selection of a content such as greater than 9.8×10 ⁸ to about 1.02×10 ⁹ white blood cells.

Other examples of selectable ranges for content or concentration embodiments include a range of white blood cells from greater than 6 x 10 ⁸ to about 2 x 10 ⁹ , preferably about 1.2 x 10 ⁹ ( including all dosage values therebetween). Another example is the selection of a range of white blood cells from greater than 7×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 8×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 8.5 x ¹⁰⁸ to about 2 x ¹⁰⁹ , preferably to about 1.2 x ¹⁰⁹ , including all dosage values therebetween. is. Another example is the selection of a range of white blood cells from greater than 9×10 ⁸ to about 2×10 ⁹ , preferably up to about 1.2×10 ⁹ , including all dosage values therebetween. . Another example is the selection of a range of white blood cells from greater than 9.5 x ¹⁰⁸ to about 2 x ¹⁰⁹ , preferably to about 1.2 x ¹⁰⁹ , including all dosage values therebetween. is. Thus, all dosage ranges that can be selected from the values herein as understood by one of ordinary skill in the art are encompassed by the present invention.

Methods for measuring each of the above cell populations will be clearly established in the detailed description of the invention. Specifically, methods for measuring each of lymphocytes, monocytes, granulocytes, platelets and RBCs are performed using total cell counts and differentiated cell counts with an automated blood cell counter (ABX Pentra 60, Horiba Medical). The percentage of progenitor cells, the percentage of CXCR4-expressing cells and the percentage of VEGFR2-expressing cells were determined using a flow cytometer (MACS Quant Analyzer 10, Miltenyi Biotec).

Note that such cell suspensions may contain pharmaceutically acceptable carriers and/or excipients.

In another preferred embodiment, the cells of the cell suspension of the invention are suspended in a volume of 5 ml to 30 ml of heparinized saline solution or lactated Ringer's solution, preferably containing about 1% HSA and about 2.5% glucose. become muddy.

The present invention also provides a pharmaceutical composition comprising a cell suspension of the invention as defined above and optionally a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may include a medium that supports cell viability and functionality. Such media can be serum-free to avoid eliciting an immune response in the recipient. The carrier is buffered and pyrogen-free.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions and/or dispersion media. The use of such carriers and diluents is known in the art. The solution is sterile and of sufficiently low viscosity to allow the use of syringes for administration.

Examples of materials and solutions that can serve as pharmaceutically acceptable carriers are also known in the art.

Note that in a preferred embodiment, the final pharmaceutical formulation solution for the cell suspension products described in the Examples herein consisted of heparinized saline (see Table 4). . Lactated Ringer's solution (2.7 mg/100 mL calcium chloride dihydrate, 320 mg/100 mL sodium lactate, 40 mg/100 mL potassium chloride and 600 mg/100 mL sodium chloride) supplemented with 2.5% glucose and 1% HSA can also be used (see Table 2).

The final formulated media described in the Examples of the present invention was prepared for each batch in a Class A BSC by adding 10 mL glucose (2.5%) and 1 mL HSA (20%) to 9 mL lactated Ringer's solution. Prepared fresh on the day of treatment.

A particularly preferred excipient is therefore lactated Ringer's, preferably supplemented with glucose and HSA.

In another preferred embodiment, the cell suspension of the invention or the pharmaceutical composition of the invention can be frozen in freezing medium. Any medium that maintains cell viability at temperatures between -135°C and -190°C is suitable as a freezing medium. For example, freezing media can contain 2.5% to 10% DMSO. More specifically, the freezing medium may contain 5%-10% DMSO.

Freezing medium is based on the culture medium or growth medium described herein and further comprises human serum or any other protein or mixture of proteins capable of maintaining cell integrity after cell thawing. You can stay.

After thawing, the cells of the invention can be washed to remove DMSO or other freezing medium components prior to dosing or resuspension in dosing solutions. A dosing solution is any physiological solution that can be injected into a patient without toxicity. Dosing solutions may contain 1% to 15% protein, eg, human serum albumin.

The pharmaceutical compositions of the invention can also be used in any of the methods of treatment or therapeutic uses described herein.

As mentioned above, the cell suspension of the present invention accelerates the (re)generation of new and existing vessels and improves blood flow within existing vessels, as illustrated in Example 2. . Therefore, the cell suspension of the present invention is useful for treating peripheral arterial disease of the lower extremities (PAD), preferably CLI, more preferably CLI with DM in patients without the option of bypass surgery or revascularization using endovascular therapy. is particularly advantageous for the treatment of Example 2 was conducted by using a specific medicinal cell suspension product included in the definition of cell suspension of the invention as defined in the first aspect of the invention. Such medicinal cell suspension products were made from BM-MNC drug substance formulated in 10 mL to 30 mL of heparinized saline. Various dosages of such medicinal cell suspension products were used, with a preferred dosage being from about 5×10 ⁸ to about 1×10 ⁹ . The final containers used were 1-3 dosing syringes. Data on this cell suspension product were collected in a Phase 2 study (CMMo/ICPD/2008) on 32 subject batches. Table 2 below summarizes the automated cell counter data for each subject, including WBC and differentiation (lymphocyte, monocyte) counts. Product-related impurities, including RBC and platelet concentrations, and granulocyte percentages are also listed for each subject.

Phenotypic analysis of the same products is also shown in Table 3, including analysis of the following cell types:
CD34+ hematopoietic stem cells CD34+CD38- early uncommitted HSC
CD133+ cell population containing endothelial progenitor cells CD90+ early hematopoietic stem cells CXCR4+ cells expressing SDF-1 receptor VEGFR2+ cells expressing vascular endothelial growth factor receptor 2 CD31+CD146+CD133- mature endothelial cells CD34+VEGFR2+CD133+ late-proliferation endothelial progenitor cells CD34-CD105+CD90+CD73+ MSC

Data collected from the drug substance are presented in Tables 2 and 3.

Based on the results collected in a Phase 2 study (CMMo/ICPD/2008) on 32 subject batches, illustrated in Tables 2 and 3 above, the cell suspension products of the present invention were characterized. gone.

Note that the invention further encompasses 1-3 (pre-filled) syringes containing the cell suspension of the invention.

Additionally, if desired, the cells of the cell suspensions of the present invention may be processed by any conventional method, including, but not limited to, transgenetic, deletion or insertional processes in their genome. It may be genetically engineered.

Methods of Making Cell Suspension Products The cell suspension products of the present invention can be made by a number of methods, the methods of manufacture include plasma, platelets, RBCs and granules. The cell suspension of the present invention is minimally engineered BM-MNC (bone marrow-mononuclear cell ) preferably prepared as detailed in Example 1 so that it can be considered as a product.

The manufacturing method described in Example 1 is briefly summarized as follows:
Step 0. Bone marrow (BM) harvesting is performed by repeated puncture, preferably from the subject's posterior iliac crest under local anesthesia. The BM puncture fluid is then collected in a shipping bag, preferably containing an acid citrate dextrose solution A (ACD-A) anticoagulant.
Step 1: Filtration—The BM is preferably filtered by gravity to remove all small bone particles and prevent blockage in subsequent steps.
Step 2: SmartRedux volume reduction - initial BM volume, preferably by using the SmartRedux program with a Sepax 2.0 device and the accompanying sterile disposable SmartRedux kit (CS-490.1), according to the manufacturer's instructions Reduce to about 50-100 mL. This step, including removal of plasma and red blood cells (RBCs), yielding approximately 50-100 mL of buffy coat product also contributes to the purity of the final product.
The volume reduction step is performed once or twice depending on the volume of starting material to be processed. BM samples up to 220 mL in volume are processed in a single cycle, and samples over 220 mL are processed in two cycles using the same kit.
For both single and two-cycle volume reduction, set the final volume to approximately 50 mL to 100 mL.
Steps 3 and 4: NeatCell Density Gradient—Sterile intermediate sample bags containing reduced volume BM are subjected to density gradient centrifugation followed by preferably 2%-4% HSA in saline solution (both pharmaceutical Two washes of the mononuclear cell (MNC) suspension are performed with a wash solution consisting of: Collect approximately 45 mL of BM-MNC product into an output bag and remove other components into a waste bag.
Step 5: BM-MNC Filtration—BM-MNC are harvested from the product bag using a 50 mL syringe and filtered through a 50 μm filter into sterile Falcon tubes. Rinse the product bag with a wash solution and filter to improve MNC recovery. Adjust final volume of BM-MNC drug substance to 40 mL-60 mL.

The final step (step 6) consists of preparing the final cell suspension product of the invention. Such final products are administered on the basis of viable leukocytes, which include granulocytes as well as the MNC fraction. Since MNC is the active ingredient, the percentage of MNC in the final product is considered a quality attribute. In this sense, there are several process parameters that can influence the quality attributes of the final product. In this regard, Table 5 shows the steps that can affect the percentage of MNC in the final product, which are considered important to the production of the product.

SmartRedux Step—This step is intended to reduce the initial BM volume to a volume small enough to be processed with the NeatCell procedure (NeatCell maximum input volume is 120 mL). Additionally, the SmartRedux process includes fractionation into RBC, plasma and buffy coat products, contributing to the purity of the NeatCell product.

The critical parameters associated with this process are the centrifugation speed and time, as they directly affect the recovery of MNCs from the BM aspirate.

NeatCell process—The automated MNC enrichment method used is designed and standardized by the manufacturer and is intended to enrich the BM aspirates for MNCs and is therefore important. The Hct of the NeatCell starting material is an important material property as it affects the efficiency of MNC purification. Critical process parameters, including centrifugation speed and time, and Ficoll density and volume, are constant in the NeatCell program.

In the MNC compartment, multiple cell populations are thought to play an important role in the mode of action of the final product. Therefore, the presence and functionality of these cells are quality attributes (CQAs) considered.

The MNC fraction obtained after Ficoll density gradient centrifugation has a lower density than Ficoll and therefore contains cells that are not dense enough to penetrate the Ficoll layer during centrifugation. As a result, the density of Ficoll used in this method is considered an important process parameter as it directly affects the composition of the MNC population. The density used is 1.077 gr/ml.

Manufacturing is a continuous process and does not involve any activation steps or other large-scale manipulations of cells that could affect the functionality of specific cell populations in the MNC fraction. Processing time can affect the functionality of the cells, therefore waiting times are not built into the process and the entire process is preferably completed within 4-5 hours in a clean room environment. Functionality can also be affected by the subject's disease state and the subject's systemic physiological variations.

Based on the above, a further aspect of the invention refers to a method for producing a cell suspension of the invention, comprising:
1. Bone marrow (BM) harvesting is performed by repeated punctures, preferably from the subject's posterior iliac crest under local or general anesthesia. The BM puncture fluid is then collected in a shipping bag, preferably containing an anticoagulant solution, more preferably an acid citrate dextrose solution A (ACD-A).
2. The BM is preferably filtered by gravity to remove all small bone fragments and prevent blockage in subsequent steps.
3. The initial BM volume is preferably reduced to about 50-100 mL, preferably by using the SmartRedux program with the Sepax 2.0 device and the accompanying sterile disposable SmartRedux kit (CS-490.1). This step, including removal of plasma and red blood cells (RBCs), which preferably yields a buffy coat product of about 50-100 mL, also contributes to the purity of the final product.
4. Optionally, the volume reduction step is performed in one or two cycles depending on the volume of starting material to be processed. BM samples up to 220 mL in volume are processed in a single cycle, and samples over 220 mL are processed in two cycles using the same kit. It is preferred to set the final volume to about 50 mL to 100 mL for both single and two cycle volume reduction.
5. The sterile intermediate sample bag containing the reduced volume BM established in step 3 or step 4 is subjected to density gradient centrifugation followed by preferably 2%-4% HSA in saline (both pharmaceutical grade). ) of the mononuclear cell (MNC) suspension with wash solutions consisting of: Collect approximately 45 mL of BM-MNC product into an output bag and remove other components into a waste bag. More preferably, the sterile intermediate sample bag containing the reduced volume BM is subjected to NeatCell density gradient centrifugation followed by preferably 2%-4% HSA in saline (both pharmaceutical grade). Two washes of the mononuclear cell (MNC) suspension are performed using wash solution. Collect approximately 45 mL of BM-MNC product into an output bag and remove other components into a waste bag.
6. The BM-MNCs are preferably withdrawn from the product bag using a 50 mL syringe and filtered into a sterile Falcon tube, preferably through a 50 μm filter. Rinse the product bag with a wash solution and filter to improve MNC recovery. Adjust final volume of BM-MNC drug substance to 40 mL-60 mL.

It is also important to note that the production of the cell suspensions of the present invention is preferably a continuous process involving enrichment of mononuclear cells and the process does not include reprocessing options.

Following drug substance preparation, MNCs are preferably centrifuged at 760 xg for 10 minutes at room temperature. heparinized saline in which the cells were transferred under sterile conditions to the final formulation (preferably supplemented with 2.5% glucose and 1% HSA and sterile non pyrogenic plastic syringe(s)) or Resuspend in lactated Ringer's solution).

Of the final product, 2-3 mL may optionally be transferred to vials for quality control (QC) testing, and preferably 8-12 mL is transferred to each sterile pyrogen-free plastic syringe under sterile conditions. The syringes, with closed cones to prevent contamination, are then packaged in sterile single-use plastic zipper bags and shipping boxes for transportation to the associated imaging-assisted therapy unit for administration to the subject.

The outline of the manufacturing method is shown in the drawings.

Applicability of the Cell Suspension Product From the results clearly shown in Example 2, in the first 12 months after administration of the cell suspension of the invention, only 2 (13%) subjects A clinically relevant response demonstrated by a Rutherford Group 1-3 (Grade 1) reduction in classification (29 (64%) subjects) compared favorably with the control group in which the body responded was shown to have achieved

At the baseline visit, 25 (56%) subjects in the treatment group who received infusions of the cell suspension of the present invention exhibited non-healing ischemic ulcers, but at the follow-up visit 12 months after dosing. By 18 (75%) subjects no longer had ulcers (1 major amputation) and 24 (60%) subjects were reliable predictors of healing. TcPO ₂ ≧40 mmHg. Angiogenesis was assessed 6 months after administration of the cell suspension of the present invention. In subjects treated with the cell suspension of the present invention, angiogenesis occurred in 26 (62%) subjects, 7, 8 and 9 in the lowest, medium and highest dose groups, respectively. seen by subject.

No subjects had Suspected Unexpected Serious Adverse Reaction (SUSAR) during treatment during follow-up. Three AEs were considered route-related and were minor injection site reactions. None of the subjects prematurely discontinued the study as a result of an AE, and no subjects experienced any AEs associated with the cell suspension of the present invention. No SAEs occurred prior to administration of Rexmyelocel-T and no SAEs occurred during the first 24 hours after administration of Rexmyelocel-T. 17 SAEs in the control group (9 subjects) and 24 SAEs in the Rexmyelocel-T treatment group (14 subjects; low dose 1×10 ⁸ BM-MNC: 4 subjects; Medium dose 5× ¹⁰ BM-MNC: 4 subjects; Highest dose 1×10 BM-MNC: ⁶ subjects) total 41 SAEs were observed during the follow-up period (12 months). was recorded by 23 subjects during Two subjects died as a result of cardiovascular disease.

Therefore, the cell suspension of the present invention is useful for treating peripheral arterial disease of the lower extremities (PAD), preferably CLI, more preferably CLI with DM in patients without the option of bypass surgery or revascularization using endovascular therapy. It is clear that it is particularly advantageous for the treatment of

Accordingly, in a further aspect of the invention, a cell suspension as defined in the section entitled "Cell Suspension Products" for use in treating or ameliorating lower extremity peripheral artery disease, preferably in a human subject. Reference is made to liquids, pre-filled syringes or pharmaceutical compositions.

Preferably, the lower extremity peripheral arterial disease is critical lower extremity ischemia.

More preferably, the cell suspension, pre-filled syringe or pharmaceutical composition defined in the section entitled "Cell Suspension Products" comprises an inflatable balloon in the distal femoral or popliteal artery of an occlusive vascular lesion. It is used as a method for treating lower extremity peripheral arterial disease, preferably severe lower extremity ischemia, by intraarterial administration to obtain a low-pressure blood flow of up to 4 atmospheres by injecting the cell suspension into the artery by placing it proximally. be.

Example 1. Materials and Methods1. CELL SUSPENSIONS AND METHODS FOR MAKING SAME To illustrate the invention, cell suspensions according to the invention made with autologous cell suspensions of BM-MNCs composed of several mature cell types and hematopoietic progenitor cells. was used in the examples below. Final product formulation was based on the number of viable WBCs present.

Characterization of the cell suspensions of the present invention was performed using total and differentiated cell counts with an automated blood cell counter (ABX Pentra 60, Horiba Medical). The percentage of progenitor cells and WBCs expressing the migratory factor SDF-1 receptor CXCR4 (CD45+ cells) or WBCs expressing the angiogenic factor VEGF receptor 2 were analyzed with a flow cytometer (MACS Quant Analyzer 10, Miltenyi Biotec). determined using

Concentrations of white blood cells (WBCs), percentages of lymphocytes, monocytes, granulocytes and progenitor cells, and total WBCs expressing CXCR4 or VEGFR2 of the 32 clinical batches are presented in Table 6.

Cell suspensions of the invention were manufactured under GMP conditions. All process steps were performed in a Class B cleanroom, semi-closed operations (spike and luer lock connections) and open operations were performed in a Class A BSC.

Briefly, the bone marrow was sterile filtered to remove bone debris, followed by an automated volume reduction step to obtain a sufficiently small volume for further processing.

Cells were then subjected to automated Ficoll density gradient centrifugation with a wash step. A BM-MNC fraction of 50 mL was obtained by filtering the product, which was used as the drug substance.

The entire drug substance manufacturing process was closed except for the spike and luer lock connections. Cell separation steps were performed in an automated system to ensure standardization of the method.

A flow diagram of the method for producing a cell suspension of the present invention is presented in FIG. Additionally, the method of manufacture is detailed below:
Step 0: Bone marrow (BM) harvesting was performed by repeated punctures from the subject's posterior iliac crest under local anesthesia using a 5 mL syringe connected to a BM lancing device. BM puncture fluid was collected into a 600 mL shipping bag containing acid citrate dextrose solution A (ACD-A) anticoagulant until a volume of approximately 250 mL to 350 mL was reached. Pack the shipping bag containing the fresh BM according to the instructions and prepare for shipment. BM is shipped at room temperature (15° C.-21° C.) to Rexgenero in one shipment for immediate processing.
Step 1: Filtration—The BM was gravity filtered through a 200 μm in-line filter to remove all small bone fragments and prevent blockage in subsequent steps. After filtration, samples were taken for in-process testing and the collection bag was weighed and mass converted to volume to obtain the input volume in the cell processing setting.
Step 2: SmartRedux volume reduction - using an initial BM volume of approximately 300 mL with the SmartRedux program on the Sepax 2.0 device and the accompanying sterile disposable SmartRedux kit (CS-490.1) according to the manufacturer's instructions was reduced to 50 mL by This step, including removal of plasma and red blood cells (RBCs), yielding a 50 mL buffy coat product also contributes to the purity of the final product. The volume reduction step was performed once or twice depending on the volume of starting material to be processed. BM samples with a volume of up to 220 mL were processed in a single cycle, and samples over 220 mL were processed in two cycles using the same kit. The final volume was set to 50 mL for both single and two cycle reductions.
Steps 3 and 4: NeatCell Density Gradient—A sterile intermediate sample bag containing reduced volume BM was connected to the Neatcell kit (CS-900.2) and subjected to a Sepax 2.0 device according to the manufacturer's instructions. It was processed using the NeatCell program. This process involves density gradient centrifugation on Ficoll 1077 followed by mononuclear cell (MNC) suspension using a wash solution composed of 2.5% or 4% HSA (both pharmaceutical grade) in saline solution. It consisted of two washes of turbidity. Approximately 45 mL of BM-MNC product was collected into the output bag and other components were removed into the waste bag.
Step 5: BM-MNC Filtration—BM-MNC were harvested from the product bag using a 50 mL syringe and filtered through a 50 μm filter into sterile Falcon tubes. MNC recovery was improved by rinsing the product bag with a wash solution and filtering. The final volume of BM-MNC drug substance was adjusted to 50 mL.

2. Selection of Study Population Mean age 64.3 (±9.5) years (range: 46-80 years) Rutherford grade II (group 4) or grade III (group 4) who are unable to undergo surgical or endovascular revascularization. A total of 60 subjects (50 males (83%) and 10 females (17%)) with CLI and DM (group 5) were enrolled and randomly assigned in a 1:1:1:1 ratio, A single dose of the cell suspension of the present invention is administered in addition to standard care (one of three dose levels: 1 x 10 ⁸ , 5 x 10 ⁸ or 1 x ¹⁰ BM-MNC 15 subjects per treatment arm) or were treated according to standard of care alone (15 subjects). The cell suspension of the invention was administered intra-arterially in a single injection. For this study, stratified randomization was applied and Rutherford CLI degree II with no option for endovascular or surgical revascularization according to the Transatlantic Inter-Society Consensus Document on Management of Peripheral Arterial Disease (TASC II). and III degrees (groups 4 and 5). If CLI is present in both legs, inject the leg determined by the attending physician to have more advanced/severe disease, unless the leg above the toes has already undergone surgical amputation. gone. Study subjects were recruited at the baseline visit (Visit 1), 24 hours and 1 month, 3 months, 6 months, 9 months, and 12 months after administration of Rexmyelocel-T (Visits 3, 4, 4, and 5, respectively). Evaluations were made at Visit 5, Visit 6, Visit 7 and Visit 8). Subjects assigned to the control group did not have to come to Visits 2 and 3.

3. Inclusion Criteria Subjects were eligible for study entry if they met all of the following inclusion criteria:
1. Male and female subjects aged 18 years or older and 80 years or older.
2. Subjects undergoing treatment for Type I or Type II DM.
3. CLI grades II and III according to Rutherford (Groups 4 and 5).
4. Inability to undergo surgical or endovascular revascularization as recommended by TASC II.
5. Life expectancy over 2 years.
6. No anticipation of major limb amputation within 12 months after enrollment.
7. Normal as defined by leukocytes ≧3000/mL, neutrophils ≧1500/mL, platelets ≧100,000/mm ³ , AST/ALT ≦2.5 times normal range, creatinine ≦2.5 mg/dL biochemical parameters.
8. The subject has provided written informed consent to participate in the study.
9. Women of childbearing potential must have a negative pregnancy test result according to each hospital's standard procedure at the time of study entry and must commit to using medically approved contraception during the study.

4. Exclusion Criteria Subjects were excluded from study participation if they met any of the following exclusion criteria:
1. History of neoplasm or hematologic disease (myeloproliferative disease, leukemia or myelodysplastic syndrome).
2. Subjects with uncontrolled hypertension (defined as blood pressure greater than 180 mmHg/110 mmHg on two or more occasions).
3. Severe heart failure (New York Heart Association stage IV).
4. A subject with malignant ventricular arrhythmia or unstable angina.
5. Diagnosis of deep vein thrombosis within 3 months prior to screening.
6. Active infection or gangrene seen on the same day that Rexmyelocel-T administration is planned.
7. Combination therapy including hyperbaric oxygen therapy, vasoactive substances, Cox-II inhibitors.
8. BMI>40 kg/ ^m2 .
9. Subjects with a diagnosis of alcoholism at enrollment.
10. proliferative retinopathy.
11. Complications that reduce life expectancy to less than 1 year.
12. HIV infection, hepatitis B or hepatitis C.
13. Subjects who were unwilling or unable to comply with all aspects of the protocol.
14. Heart failure or an ejection fraction less than 30%.
15. Stroke or myocardial infarction within 3 months prior to the screening visit.
16. Anemia (hemoglobin <10 g/dL).
17. Leukopenia.
18. Thrombocytopenia (platelets less than 100000/mm ³ ).
19. Women of childbearing age who were pregnant or were not using adequate contraception.
20. Subjects who had participated in a clinical trial within 3 months prior to screening for this study.

5. Exclusion of Subjects from the Study Subjects could be excluded from the study for any of the following reasons:
Inability to achieve optimal final concentration of cell suspension based on randomization results. In this case, the PI should decide whether to administer the product.
Presence of serious adverse events (SAEs) from informed consent to planned dosing date.
Big protocol deviation. Post-randomization discovery that a subject is unable to meet protocol entry criteria or does not adhere to protocol requirements and that continued participation poses an unacceptable risk to the subject's health.
The subject had an AE or pre-treatment event that required early termination because continued participation would pose an unacceptable risk to the subject's health, or the subject wished to continue because of the AE or pre-treatment event. I didn't.
Voluntary withdrawal. Subject (or subject's legal representative, etc.) wishes to withdraw from the study.
Untraceable. The subject did not return to the clinic and attempts to contact the subject were unsuccessful. Attempts to contact subject were documented. If a subject withdrew from the study, all procedures scheduled for an early termination visit were performed, if possible.

Alternative therapy was recommended for all subjects prematurely withdrawn from the study as per good clinical practice of pharmaceuticals. If the withdrawal is due to the development of a significant AE (adverse event), the subject will be monitored by the principal investigator until completion, i.e., until the AE resolves or until it is determined that it is a permanent event. rice field. The primary reason for study discontinuation was recorded on the CRF.

6. Treatment Study Medication Performed Under local anesthesia and sedation, a volume of BM (90 mL-420 mL) was collected from the iliac crest by serial puncture. BM was collected in a transport bag containing ACD-A solution as anticoagulant at a ratio of 1:5 (BM volume) (see item 1 above).

Suspensions of BM-MNC were obtained by density gradient centrifugation without modification or addition of any products that might affect their biological functionality.

The cell suspension of the invention was administered in the following single doses:
1×10 ⁸ BM-MNCs
5×10 ⁸ BM-MNCs
1×10 ⁹ BM-MNCs

The cell suspension of the invention was administered using an intra-arterial catheter. The artery of the target limb is intubated by a transfemoral approach with an over-the-wire catheter balloon (matching the popliteal diameter) advanced as distally as possible but not below the knee in all cases, usually in the distal thigh or knee. It was placed proximal to the obstructive vascular lesion at the foveal artery. At this point, the balloon was inflated to block blood flow and the cell suspension of the invention was slowly infused over 3 minutes (min). After administration, the balloon was deflated to restore anterograde blood flow.

Administration of the cell suspension of the present invention was performed 3-5 hours after BM harvesting by a vascular specialist (imaging physician or vascular surgeon) dispatched directly from the cell therapy unit.

All subjects were treated with the current standard of care for subpopliteal severe atherovascular disease as described in the TASC II treatment guidelines.

7. Changes in the Rutherford Classification Critical limb ischemia includes patients with leg rest pain, ischemic ulcers or gangrene secondary to severely impaired blood flow in the affected limb that persists for more than 2 weeks. Subjects' PAD classification was vascular, using grades rather than Rutherford's categories because they did not undergo a standard treadmill exercise test and could not formally distinguish between mild, moderate, or severe IC. It was done by specialist examination.

IC (Grade I, Groups 1-3) is extremity induced by exercise (caused by walking or muscle activity) and relieved immediately upon cessation of activity, unlike persistent resting pain in patients with CLI muscle pain, discomfort or weakness.

Rutherford's degree 0 was used to identify subjects who had no symptoms or only cold sensations, no clinical signs of obstructive disease, or slight hypocardia.

A non-healing ischemic ulcer suggests that there is inadequate arterial perfusion of the leg to support the inflammatory response that requires healing. Associated with this is usually ischemic rest pain and objective evidence of diffuse leg ischemia (CLI Rutherford grades III-IV, groups 5-6).

Ischemic resting pain indicates diffuse paw ischemia (degrees II-IV, groups 4-6) and cannot be easily controlled by analgesics. Pain is localized in the forelimbs and toes, or around focal ischemic lesions. Pain is aggravated by elevating the limb. Diffuse foot ischemia with ischemic resting pain is commonly associated with ankle blood pressure <40 mmHg and ABI <0.8.

Subjects were classified based on the presence of ischemic rest pain (with or without non-healing ischemic ulcer), exercise-induced ischemic myalgia (IC), or asymptomatic disease. To classify study subjects, clinical symptoms were combined with objective evidence of diffuse foot ischemia by measuring ABI (if it can be reliably assessed).

Changes in Rutherford classification from baseline to 1 month, 3 months, 6 months, 9 months and 12 months were evaluated.

Clinical improvement was defined as a single Rutherford grade (category) improvement from baseline. Clinically relevant improvement was defined as a change in Rutherford classification from CLI group 4 or 5 to IC group 3 or less after 3 months, 6 months, 9 months or 12 months after administration of Rexmyelocel-T. .

8. Number of Ulcers and Changes in Ulcer Size The size of the largest ulcer on the target leg and the total number of ulcers on both legs were recorded on the Case Report Form (CRF). Changes in ulcer healing and ulcer size from baseline to 1 month, 3 months, 6 months, 9 months and 12 months were evaluated.

9. Angiogenesis Angiogenesis was measured 6 months after administration of the cell suspension of the present invention comparing baseline angiography performed before treatment with angiography performed 6 months after administration of Rexmyelocel-T. It was evaluated by an evaluation panel consisting of two imaging treating physicians who performed an independent, blinded evaluation by doing.

Longitudinal growth of existing arteries, cross growth of existing arteries, increased number of collateral vessels, increased collateral vessel size and/or density, or major subpopliteal vessel size and/or density Angiogenesis is considered to be occurring when there is an increase in .

10. Major amputation (target limb)
A major amputation was considered an amputation of the lower extremity above the ankle.

11. ABI Change ABI is defined as the ratio of the systolic pressure measured at the ankle to the systolic pressure measured at the brachial artery. Subjects were instructed to rest in a supine position with head and heel support for 5-10 minutes in a room at a temperature of 19-22°C. Appropriately sized blood pressure cuffs were selected for both ankle and arm blood pressure measurements. For both the ankle and arm, the cuff was wrapped around the limb using a straight wrapping technique. An 8-10 MHz Doppler probe was used and Doppler gel was applied onto the sensor. A Doppler probe was also used to detect brachial blood flow during arm blood pressure measurements. Brachial systolic pressures were measured in both arms, as well as anterior and posterior tibial systolic pressures in the limbs in question.

Changes in ABI from baseline to 1, 3, 6, 9 and 12 months were recorded in subjects who were able to reliably assess ankle blood pressure.

12. Changes in transcutaneous oxygen partial pressure Transcutaneous oximetry _TcPO2 is a local non-invasive measurement that reflects the amount of _O2 diffused through the epidermis from capillaries. _TcPO2 was measured using TCM3 (Radiometer Medical ApS, Copenhagen, Denmark).

Changes in _TcPO2 from baseline to 1 month, 3 months, 6 months, 9 months and 12 months were recorded.

13. Adverse Event Definition An adverse event (AE) was defined as any untoward experience experienced by a subject during the study, whether or not considered related to study drug. Therefore, an AE is defined as any unfavorable unintended manifestation (including abnormal laboratory findings) associated with the use of the medicinal product (investigational product) over time, whether or not it was related to the medicinal product (study product). , a symptom or a disease. This includes exacerbation of a pre-existing condition or event, co-occurring illness, drug-drug interactions, or significant deterioration of an investigational indication not documented anywhere in the case report under the specified efficacy assessment. It was Expected changes in pre-existing conditions, including diseases under study that did not show clinically significant exacerbation or exacerbation, were not considered AEs.

The following data were documented for each AE:
A description of the symptom event.
Classification as “serious” or “not serious”.
Severity: Mild (AEs easily tolerated by subject; cause minor discomfort and do not interfere with daily activities), Moderate (AEs unpleasant enough to interfere with normal daily activities; intervention indicated) severe (AE that interferes with normal daily activities; treatment or other intervention is usually required).
Date of first occurrence and date of resolution (if applicable).
Action taken: Any action, permanent discontinuation of study treatment, administration of concomitant medications, administration of non-pharmacological treatment or admission/long-term admission (SAE).
Causal relationship. Every effort was made by the principal investigator to assess the relationship between AEs and Rexmyelocel-T, if any. Causality was assessed using the following categories:
If there was little evidence to suggest causality (e.g., the event did not occur within a reasonable period of time after administration of Rexmyelocel-T, or another plausible explanation for the event (e.g., subject's clinical condition, other concomitant treatment)), unlikely. If there is evidence suggesting a possible causal relationship (e.g., other factors (e.g., subject's clinical condition, other concomitant treatments) may have contributed to the event, but the event occurred after administration of Rexmyelocel-T). occurred in a reasonable amount of time), it is possible. Likely when there is evidence suggesting causation and the influence of other factors is unlikely.
It is certain when there is clear evidence to suggest causation and the possible contribution of other factors can be ruled out.
It is unreadable when there is insufficient or incomplete evidence to make a clinical judgment about causation.
Event outcome (unknown, recovered, not recovered, recovered with sequelae, died (report date and cause)).

14. Definition of Serious Adverse Event An SAE was defined as an AE that met one or more of the following serious outcome criteria:
caused death.
It was deadly.
Inpatient hospitalization or extension of existing hospitalization was required.
Caused permanent or serious disability or incapacity.
Congenital anomalies or birth defects.
Based on medical and scientific judgment, it was a significant medical event that had the potential to endanger the subject or required intervention to prevent one of the outcomes listed above.

An AE was considered fatal if the subject was at risk of death at the time of the event. It was not considered an event that could theoretically have caused death if it had been more severe.

Example 2. Results 1. Changes in Rutherford Classification Changes in Rutherford categories of PAD in control subjects and subjects treated with a cell suspension of the invention at baseline and after 3, 6, 9 and 12 months are shown below. Presented in Table 7.

At the baseline visit, subjects in the cell suspension treatment arm of the present invention were either CLI Rutherford Group 4 (degree II) or Rutherford Group 5 (degree III) (20 (44%) subjects and 25 subjects, respectively). (56% of subjects). Results showed that during the first 12 months, the majority of subjects treated with the cell suspension of the present invention improved, namely Rutherford Groups 0-3 (0 and 1 degrees) in 28 (68%) subjects. demonstrated a clinically relevant response demonstrated by a reduction in classification to

After 3 months of dosing, 15 (33%) subjects treated with the cell suspension of the invention and 12 (40%) subjects in the medium and high dose groups no longer had pain at rest. or did not suffer tissue loss and were classified as Rutherford Groups 0-3 (degrees 0 and 1), which accounted for 21 (46%) subjects at 6, 9 and 12 months respectively after dosing. , further increased to 24 (57%) and 28 (68%). For subjects in the medium and high dose groups, this was 13 (43%), 15 (50%) and 18 (64%) after 6, 9 and 12 months of dosing, respectively. there were. After 3, 6, 9 and 12 months, respectively, 0 (0%), 1 (8%), 1 (8%) and 2 (18%) subjects It was compared favorably with the control group, which improved from Groups 1 to 3 (degree I).

1 subject assigned to the control group withdrew informed consent after randomization, 1 subject died of cardiovascular disease before the 6-month follow-up visit, and 1 patient died at the 6-month follow-up visit. One subject underwent a major amputation prior to the follow-up visit and was lost to follow-up before the 12-month visit. Two subjects given the lower dose of Rexmyelocel-T underwent major amputation of the target limb 6 months after dosing.

9 of 11 subjects (82%) reported rest pain or the presence of non-healing ischemic ulcers (4 (36%) subjects) in comparison to the control group. A total of 28 (62%) of 45 subjects in the cell suspension treatment group (18 of 30 (63%) in the medium and high dose groups) were at rest after 12 months of dosing. No ischemic pain or ischemic ulcers were noted.

When last observation carried forward (LOCF) was applied (excluding subjects who underwent major limb amputation; these subjects were considered non-responders), the results In the first 12 months after administration of the cell suspension of the invention, 28 (64%) of all treated subjects and 18 (63%) in the medium and high dose groups had a Rutherford 0 to It was shown that clinically relevant responses demonstrated by reduction in classification into Group 3 (0 and I degrees) were achieved (see Table 8 and Figures 2 and 3).

After 3 months of administration of Rexmyelocel-T, 15 (33%) subjects treated with Rexmyelocel-T (12 of them (40%) in the medium and high dose groups) no longer had rest pain or tissue No deficits were categorized as Rutherford Groups 0-3 (degrees 0 and 1), with 21 (46%) subjects after 6 and 9 months of dosing for all treated patients, respectively. and further increased to 24 (53%) subjects. In the medium and high dose groups, 14 (47%) and 15 (50%) subjects were classified into Rutherford groups 0-3 at 6 months and 9 months, respectively. After 3 months, 6 months, 9 months and 12 months respectively, only 0 (0%), 1 (8%), 1 (7%) and 2 (13%) subjects It compared favorably with the control group, which improved to Rutherford Groups 1-3 (degree I).

Figure 4 and Table 9 demonstrate the clinically relevant response of more subjects in the treatment group with the cell suspension of the present invention compared to the control group at all post-dose follow-up visits. By 12 months after administration of Rexmyelocel-T, the majority of subjects in all Rexmyelocel-T groups showed a clinically relevant response compared to the control group: 34 (76%) subjects body (10, 13 and 11 subjects in the lowest, medium and highest dose Rexmyelocel-T groups, respectively) vs. 2 (13%) subjects.

2. Changes in Ulcer Counts in Affected Limbs Changes in ulcer counts in affected limbs are presented in Table 10 and FIG. At the baseline visit, 25 subjects (56%) in the treatment group with cell suspensions of the invention and 9 subjects (60%) in the control group were classified as the Rutherford 5 group and had non-healing deficiencies. showed a bloody ulcer. Most of the subjects had one ulcer, and there was no significant difference between the treatment and control groups (5 subjects each in the lowest, medium and highest dose cell suspension groups of the present invention). , 7 and 6 subjects, and 6 subjects in the control group). 4 subjects (1, 1 and 2 subjects respectively) in the treatment group with the lowest, medium and highest doses of the cell suspension of the invention and 1 subject in the control group for 3 had more than one ulcer.

Mean ulcer size at baseline in subjects treated with the cell suspension of the present invention was greater than in control subjects (39.9 ± 23.0 mm vs. 26.3±23.5 mm), decreased ulcer size in subjects whose ulcers did not heal (29.7±13.5 mm), and increased mean ulcer size in controls (43.8±29.3 mm ) was shown from the results (Table 11).

3. Changes in _TcPO2 Changes in _TcPO2 are presented in Table 12.

At baseline, 35 (79%) subjects in the cell suspension treatment group of the invention had a _TcPO2 <40 mmHg compared to 6 (43%) subjects in the control group. (12, 12 and 11 subjects in 3 dose groups (lowest, medium and highest dose groups, respectively)).

Results showed that by the 12-month follow-up visit, the majority of subjects treated with Rexmyelocel-T (24 (60%) ) showed an increase in TcPO ₂ levels to above 40 mmHg.

4. Angiogenesis Angiogenesis was assessed 6 months after administration of the cell suspension of the present invention. In subjects treated with the cell suspension of the present invention, angiogenesis occurred in 26 (62%) subjects, 7, 8 and 9 in the lowest, medium and highest dose groups, respectively. seen in the subjects (see Table 13).

Angiographic images of 3 of the 24 angiogenesis events that occurred are presented relative to baseline in FIG. Parallel lateral growth (Fig. 6C).

Claims (15)

An agent for treating or improving lower extremity peripheral artery disease, which is a cell suspension containing 4×10 ⁸ to 1.2×10 ⁹ autologous or allogeneic leukocytes derived from the bone marrow of a human subject. out of the total number of white blood cells of 4×10 ⁸ to 1.2×10 ⁹ ,
i. 20%-51% are lymphocytes and 3.9%-22.3% are monocytes;
ii. 1.4% to 10% are hematopoietic stem cells expressing CD34;
iii. 25.3% to 83.3% of the total number of white blood cells are mononuclear cells;
iv. 16.7% to 74.7% are granulocytes,
v. 5.4% to 38.8% of the total number of white blood cells express CXCR4,
vi. 0.07% to 24.7% of the total number of white blood cells express VEGFR2,
vii. Of the total number of CD34-expressing hematopoietic stem cells, 7.7%-55.5% are early uncommitted hematopoietic stem cells that do not express CD38, and 0.7%-10.3% are CD34- and CXCR4-expressing stem cells. and
viii. A therapeutic or ameliorating agent comprising a cell suspension having a maximum red blood cell to leukocyte cell ratio of 6.7 and a maximum platelet to leukocyte cell ratio of 32. 20. In said cell suspension, 32.3% to 80.0% of the total number of white blood cells are mononuclear cells selected from the list consisting of lymphocytes, monocytes and hematopoietic stem cells expressing CD34; The therapeutic or improving agent according to claim 1, wherein 0% to 67.7% are granulocytes. 3. The therapeutic or ameliorating agent according to claim 1 or 2, wherein said cell suspension contains 5×10 ⁸ to 1.2×10 ⁹ autologous or allogeneic leukocytes. 3. The therapeutic or ameliorating agent according to Claim 1 or 2, wherein said cell suspension contains 8 x ¹⁰⁸ to 1.2 x ¹⁰⁹ autologous or allogeneic leukocytes. 3. The therapeutic or ameliorating agent according to claim 1 or 2, wherein said cell suspension contains 9×10 ⁸ to 1.1×10 ⁹ autologous or allogeneic leukocytes. The therapeutic or ameliorating agent according to claim 1 or 2, wherein said cell suspension contains 9.5×10 ⁸ to 1.05×10 ⁹ autologous or allogeneic leukocytes. The therapeutic or ameliorating agent according to claim 1 or 2, wherein said cell suspension contains 9.8×10 ⁸ to 1.02×10 ⁹ autologous or allogeneic leukocytes. The therapeutic or ameliorating agent according to any one of claims 1 to 7, wherein the lower extremity peripheral artery disease is critical lower extremity ischemia. of claims 1-8, wherein the cells of said cell suspension are suspended in a volume of 5 ml to 30 ml of heparinized saline or lactated Ringer's solution, preferably containing about 1% HSA and about 2.5% glucose. A therapeutic or ameliorating agent according to any one of the claims. 8. A therapeutic or ameliorating agent as defined in any one of claims 1-7, wherein an inflatable balloon is positioned proximal to the occlusive vascular lesion in the distal femoral or popliteal artery, and the cell suspension A therapeutic or ameliorating agent for use in a method of treating lower extremity peripheral arterial disease, preferably severe lower extremity ischemia, by intra-arterial administration to obtain a low-pressure blood flow of up to 4 atmospheres by injecting into an artery. A syringe or syringes containing a therapeutic or ameliorating agent as defined in any one of claims 1-7. 12. The syringe according to claim 11, which is used for treating or improving lower extremity peripheral artery disease, preferably critical lower extremity ischemia. An inflatable balloon is placed in the distal femoral or popliteal artery proximal to the occlusive vascular lesion and the cell suspension is infused intra-arterially to obtain low-pressure blood flow up to 4 atmospheres. 12. A syringe according to claim 11 for use in a method of treating arterial disease, preferably critical limb ischemia. A therapeutic or ameliorating agent according to any one of claims 1 to 7 or a syringe for use according to claims 12 or 13, wherein said suspension is provided as a single dose. Therapeutic or ameliorating agent or syringe for use according to claim 10 or 13, wherein induction of low pressure blood flow is for 1 to 6 minutes and infusion of said cell suspension is for 2 to 10 minutes.

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